The present invention relates generally to telomeric binding proteins, in particular to telomeric repeat binding factors (TRFs), to the nucleotide and amino acid sequences encoding the TRFs, and to diagnostic and therapeutic methods of use thereof. TRFs have particular uses in the treatment of cancer and aging.
Eucaryotic chromosomes end in specialized structures, called telomeres [Muller, The Collecting Net-Woods Hole, 13:181-195 (1939] that are thought to fulfill at least three functions. First, telomeres protect natural double-stranded DNA ends from degradation, fusion, and recombination with chromosome-internal DNA [McClintock, Genetics, 26:234-282 (1941)]. Second, cytogenetic observations indicate that telomeres are located at the nuclear periphery, suggesting a role for chromosome ends in the architecture of the nucleus [Agard et al., Nature, 302:676-681 (1983); Rabl, Morphol. J., 10:214-330 (1885)]. Third, telomeres must provide a solution to the end-replication problem [Watson, Nature, 239:197-201 (1972)]: because all known polymerases require a primer and synthesize DNA from 5xe2x80x2 to 3xe2x80x2, the 3xe2x80x2 ends of linear DNA pose a problem to the replication machinery.
The single common structural feature of most eucaryotic telomeres is the presence of a tandem array of G-rich repeats which, according to genetic studies in Saccharomyces cerevisiae, are necessary and sufficient for telomere function [Lundblad et al., Cell, 83:633-643 (1989); Szostak et al., Cell, 36:459-568 (1982)]. Although all telomeres of one genome are composed of the same repeats, the terminal sequences in different species vary. For instance, Oxytricha chromosomes terminate in TTTTGGGG repeats [Klobutcher et al., Proc. Natl. Acad. Sci. USA, 78:3015-3019 (1981)], Tetrahymena utilizes an array of (TTGGGG)n [Blackburn et al., J. Mol. Biol., 120:33-53 (1978)], plant chromosomes carry the sequence (TTTAGGG)n(Richards et al., Cell, 53:127-136 (1988)], and trypanosomas and mammals have TTAGGG repeats at their chromosome ends [Blackburn et al., Cell, 36:447-458 (1984); Brown, Nature, 338:774-776 (1986); Cross et al., Nature, 338:771-774 (1989); Moyzis et al., Proc. Natl. Acad. Sci. USA, 85:6622-6626 (1988); Van der Ploeg et al., Cell 36:459-468 (1984)]. The organization of the telomeric repeats is such that the G-rich strand extends to the 3xe2x80x2 end of the chromosome. At this position, telomerase, an RNA-dependent DNA polymerase, first demonstrated in Tetrahymena thermophila and other ciliates, can elongate telomeres, probably by using an internal RNA component as template for the addition of the appropriate G-rich sequence [Greider and Blackburn, Cell, 43:405-413 (1985)]. This activity is thought to complement the inability of polymerases to replicate chromosome ends, but other mechanisms of telomere maintenance may operate as well [Pluta et al., Nature, 337:429-433 (1989)]. Recently, it has been reported that the addition of telomerase into a cultured human cell leads to an increase of the proliferative life-span of that cell [Bodner et al., Science, 279:349-352 (1998)].
Proximal to the essential telomeric repeats, some chromosome ends harbor additional common elements called sub-telomeric repeats or telomere-associated sequences [Chan et al., Cell, 33:563-573 (1983); Corcoran et al., Cell, 53:807-813 (1988); de Lange et al., Nucl. Acids. Res., 11:8149-8165 (1983); Van der Ploeg et al. (1984); Dunn et al., Cell, 39:191-201 (1984)]. Unlike telomeric repeats, these sequences are not conserved and their function remains unclear [Murray et al., Mol. Cell. Biol., 6:3166-3172 (1986)].
Chromosome ends of unicellular organisms often show structural instability. Frequent rearrangements of subtelomeric sequences occur in trypanosomas [Borst, Annu. Rev. Biochem., 55:701-732 (1986), de Lange et al. (1983)], S. cerevisiae [Carlson et al., Mol. Cell. Biol., 3:351-359 (1983); Horowitz et al., Mol. Cell. Biol., 4:2509-2517 (1984)], and plasmodia [Corcoran et al., (1988)], and changes in the telomeric repeat region can be observed in protozoa [Bernards et al., Nature, 303:592-597 (1983); Pays, Nucl. Acids. Res., 11:8137-8147 (1983); Van der Ploeg (1984)], ciliates [Larson et al., Cell, 50:477-483 (1987)], and fungi [Carson et al., Cell, 42:249-257 (1985); Lundblad et al., (1989); Lustig et al., Proc. Natl. Acad. Sci. USA, 83: 1398-1402 (1986)]. As much as 3.5 kilobase pairs (kb) was seen to be added to telomeres of Trypanosoma brucei in a process that appears gradual and continuous, and was calculated to result from the addition of 6 to 10 base pairs (bp) per end per cell division [Bernards et al., (1983); Pays et al., (1983); Van der Ploeg, (1984)]. A similar gradual telomere elongation, compatible with the addition of telomeric repeats by telomerase, occurs in continuously growing T. thermophila [Larson, (1987)] and a cell cycle mutant (cdc 17) of S. cerevisiae [Carson et al., (1985)]. In wild-type S. cerevisiae [Shampay et al., Proc. Natl. Acad. Sci. USA, 85:534-538 (1988)], however, and in T. thermophila grown in batch cultures [Larson et al., (1998)], the tandem array of telomeric repeats is maintained at constant length. At least four genes (CDC 17, EST 1, TEL1, and TEL2 [Carson et al., (1985); Lundblad et al., (1989); Lustig et al., (1986)] govern the length and stability of yeast telomeres; their mode of action is not understood.
Much less is known about the structure and behavior of chromosome ends of multicellular organisms. Mammalian telomeres have become amenable to molecular dissection with the demonstration that telomeric repeats of plants and T. thermophila species cross-hybridize to vertebrate chromosome ends [Allshire et al., Nature, 332:656-659 (1988); Richards et al., (1988)]. It has also been shown that human DNA contains tandem arrays of TTAGGG repeats, probably at the chromosome ends, providing further evidence for the evolutionary conservation of telomeres and a tool for the isolation of telomeric DNA [Moyzis et al., (1988)]. Two strategies to obtain human chromosome ends have proven successful: an indirect isolation protocol that relies on human telomeres to be functional in S. cerevisiae [Brown et al., (1989); Cross et al., (1989)] and direct cloning in E. coli. 
de Lange et al. [Mol. Cell. Biol., 10:518-527 (1990)] characterized the structure and variability of human autosomal chromosome ends. The chromosome ends they analyzed shared a sub-telomeric repeat of at least 4 kb that is not conserved in rodent genomes. These chromosome ends were characterized by a long stretch of DNA, of up to 14 kb, that lacks restriction enzyme cutting sites and may be entirely composed of TTAGGG repeats. From this region sequences are lost during development, leading to shortened, heterogeneously sized telomeres in somatic tissues, primary tumors, and most cell lines.
de Lange [EMBO J., 11:717-724 (1992)] reported that human telomeres are tightly associated with the nuclear matrix. Telomeres were demonstrated to be anchored via their TTAGGG repeats. Moreover, TTAGGG repeats at internal sites within the chromosome do not behave as matrix-attached loci, suggesting that the telomeric position of the repeats is required for their interaction with the nuclear matrix. This evidence is consistent with the role of telomeres in a nucleoprotein complex.
TRF activity was first identified in 1992 by Zhong et al. [Mol. Cell. Biol., 12:4834-4943 (1992)] as a DNA-binding factor specific for TTAGGG repeat arrays. TRF was found to be present in nuclear extracts of human, mouse and monkey cells. The optimal site for TRF binding was found to contain at least six contiguous TTAGGG repeats. However, the protein isolated by Zhong et al. was not sufficiently purified from other DNA-binding proteins such that its amino acid sequence could be determined.
Saltman et al. [Chromosoma, 102:121-128 (1993)] characterized the molecular structure of telomeres of two human tumor cell lines with frequent end-to-end associations of metaphase chromosomes. Such end-to-end associations have been observed in a variety of human tumors, aging cells and several chromosome instability syndromes. The telomeres of such end-associated chromosomes were shown by Saltman et al. to be severely reduced compared to other human cells with functional telomeres. However, other cell lines with severely shortened telomeres were not detectably compromised in their function. Thus, the investigators suggested that telomeric length was not the only determinant of the fusigenic behavior of human telomeres in tumor cells.
A Xenopus laevis protein factor that specifically recognizes vertebrate telomeric repeats at DNA ends, termed Xenopus telomere end factor (XTEF) was identified by Cardenas et al. in 1993 [Genes and Devel., 7:883-894 (1993)]. The DNA-binding properties of XTEF resembled the characteristics of a class of terminus-specific telomere proteins identified in hypotrichous ciliates.
There has been speculation on the role of an enzyme termed telomerase in human cancer, in particular in ovarian carcinoma [de Lange, Proc. Natl. Acad. Sci. USA, 91:2882-2885 (1994)]. Telomerases use the 3xe2x80x2 end of DNA as a primer and employ an RNA template for the synthesis of G-rich telomeric repeats. Telomerase activation appears to be an obligatory step in the immortalization of human cells.
Hanish et al. [Proc. Natl. Acad. Sci. USA, 91:8861-8865 (1994)] examined the requirements for the formation of human telomeres from TTAGGG seeds, and found that telomere formation was not correlated with the ability of human telomerase to elongate telomeric sequences in vitro, and did not appear to be a result of homologous recombination. Rather, the investigators reported that the sequence dependence of telomere formation matched the in vitro binding requirements for TRF1.
Although the activity of TRF1 had been identified and isolated to some extent, the purification of TRF was fraught with difficulty, both in isolating the protein away from other DNA binding proteins, and in obtaining active protein from the isolate.
Therefore, there is a need to isolate and characterize vertebrate TRF1. In addition, there is a need to identify other vertebrate telomere repeat binding factors which must also serve as structural and/or functional proteins in the maintenance of normal telomere physiological processes. Further, there is a need to isolate and characterize such TRFs (including TRF2) and to distinguish their characteristics from TRF1, as well as ascertain the role such TRFs play in telomere maintenance and elongation.
The present invention provides vertebrate telomeric binding factors (TRFs) that bind to the TTAGGG repeat sequences of telomeres. Such TRFs comprise two key domains: a dimerization domain, and a Myb domain. In addition, at least some TRFs, e.g. mammalian TRF1 and TRF2 contain a third domain, a polar N-terminal domain. In specific examples, the TRF nucleotide sequence is isolated from human, murine, or avian sources. The present invention includes these nucleic acids, the TRFs they encode, the individual domains of the encoded TRFs, and the nucleic acids that encode these individual domains.
In one particular embodiment of the present invention the TRF has the following characteristics:
a) it binds to telomere repeat sequences, in particular, TTAGGG repeats;
b) the DNA binding activity in a purified form requires the presence of another factor such as casein; and
c) it exhibits substantial sequence homology to Myb type DNA binding domains.
The present invention includes a nucleic acid or a degenerate variant thereof, which encodes a TRF of the present invention; preferably a recombinant DNA molecule or cloned gene. For example a recombinant DNA molecule or cloned gene, encodes a TRF such a TRF1 which has a nucleotide sequence of (or complementary to) SEQ ID NO: 11 (shown in FIGS. 2A-2B), or SEQ ID NO:22. These nucleotide sequences encode a TRF having an amino acid sequence of SEQ ID NO: 12 or 23 respectively which are also part of the present invention. A nucleotide sequence of a TRF 1 having a nucleotide sequence of SEQ ID NO:24 is also part of the present invention.
The present invention also provides an isolated nucleic acid encoding a vertebrate telomere repeat binding factor (TRF) which is a TRF2 having an amino acid sequence substantially homologous to that of SEQ ID NO:27, and comprising the following characteristics: a basic N-terminal domain; a dimerization domain; and a Myb domain. In one such embodiment when the basic N-terminal domain is removed the TRF detectably binds to the telomere repeat sequence (TTAGGG)12. Such binding is preferably detected in an in vitro assay.
In a preferred embodiment of this type, the isolated nucleic acid encodes a TRF that is a mammalian protein. More preferably the isolated nucleic acid encodes a human TRF having the amino acid sequence of SEQ ID NO:27, or SEQ ID NO:27 with a conservative amino acid substitution. In a particular embodiment the nucleic acid encodes a human TRF having the amino acid sequence of SEQ ID NO:27. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:26.
In another embodiment the isolated nucleic acid encodes a murine TRF having the amino acid sequence of SEQ ID NO:29, or SEQ ID NO:29 with a conservative amino acid substitution. In a particular embodiment the nucleic acid encodes a murine TRF having the amino acid sequence of SEQ ID NO:29. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:28.
Another aspect of the invention includes a nucleic acid encoding an avian TRF1 having the nucleotide sequence of SEQ ID NO:24.
All of the isolated nucleic acids of the present invention can further comprise a heterologous nucleotide sequence. Such heterologous nucleotide sequences can encode, for example, a fusion peptide (e.g., a FLAG-tag as in Example 7 below) or a chimeric protein partner such as a fusion protein. In addition any isolated nucleic acid of the present invention e.g., the corresponding recombinant DNA molecule or cloned gene can be operatively linked to an expression control sequence which may be introduced into an appropriate host. The present invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding a TRF, or a structural/functional domain of a TRF of the present invention.
In one such example, the present invention provides the isolated nucleic acid encoding a vertebrate TRF having an amino acid sequence of SEQ ID NO:27 operatively linked to an expression control sequence. The present invention also provides a unicellular host transformed or transfected with the nucleic acid. In addition the present invention provides a method of expressing the TRF encoded by the nucleic acid which comprises culturing the unicellular host in an appropriate cell culture medium under conditions that provide for expression of the protein by the cell. This method can further comprise the step of purifying the TRF. The purified form of the TRF obtained by such methodology is also part of the present invention. This methodology is intended to be general and is suitable for the expression and isolation of all of the nucleic acids of the present invention.
According to other preferred features of certain preferred embodiments of the present invention, a recombinant expression system is provided to produce biologically active vertebrate TRFs, including human TRFs as well as TRF structural/functional domains, TRF chimeric proteins and the like.
The present invention also includes nucleic acids that encode the dimerization domain of a TRF and/or the basic or acidic N-terminal domain of a TRF. In one such embodiment an isolated nucleic acid comprises a nucleotide sequence encoding a basic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:37, or SEQ ID NO:37 with a conservative amino acid substitution. In a particular embodiment the nucleic acid encodes a basic N-terminal domain that has the amino acid sequence of SEQ ID NO:37. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:36. In another such embodiment an isolated nucleic acid comprises a nucleotide sequence encoding a basic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:39, or SEQ ID NO:39 with a conservative amino acid substitution. In a particular embodiment the nucleic acid encodes a basic N-terminal domain that has the amino acid sequence of SEQ ID NO:39. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:38.
In another embodiment an isolated nucleic acid comprises a nucleotide sequence encoding an acidic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:33, or SEQ ID NO:33 with a conservative amino acid substitution. In a particular embodiment the nucleic acid encodes an acidic N-terminal domain that has the amino acid sequence of SEQ ID NO:33. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:32. In another such embodiment an isolated nucleic acid comprises a nucleotide sequence encoding an acidic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:35, or SEQ ID NO:35 with a conservative amino acid substitution. In a particular embodiment the nucleic acid encodes an acidic N-terminal domain that has the amino acid sequence of SEQ ID NO:35. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:34.
In still another embodiment an isolated nucleic acid comprises a nucleotide sequence encoding a dimerization domain of a TRF that has the amino acid sequence of SEQ ID NO:45, or SEQ ID NO:45 with a conservative amino acid substitution. In a particular embodiment the nucleic acid comprises a nucleotide sequence encoding a dimerization domain that has the amino acid sequence of SEQ ID NO:45. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:44.
In a related embodiment an isolated nucleic acid comprises a nucleotide sequence encoding a dimerization domain of a TRF that has the amino acid sequence of SEQ ID NO:47, or SEQ ID NO:47 with a conservative amino acid substitution. In a particular embodiment the nucleic acid comprises a nucleotide sequence encoding a dimerization domain that has the amino acid sequence of SEQ ID NO:47. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:46.
In another embodiment an isolated nucleic acid comprises a nucleotide sequence encoding a dimerization domain of a TRF that has the amino acid sequence of SEQ ID NO:43, or SEQ ID NO:43 with a conservative amino acid substitution. In a particular embodiment the nucleic acid comprises a nucleotide sequence encoding a dimerization domain that has the amino acid sequence of SEQ ID NO:43. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:42.
In yet another embodiment an isolated nucleic acid comprises a nucleotide sequence encoding a dimerization domain of a TRF that has the amino acid sequence of SEQ ID NO:49, or SEQ ID NO:49 with a conservative amino acid substitution. In a particular embodiment the nucleic acid comprises a nucleotide sequence encoding a dimerization domain that has the amino acid sequence of SEQ ID NO:49. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:48.
In still another embodiment an isolated nucleic acid comprises a nucleotide sequence encoding a dimerization domain of a TRF that has the amino acid sequence of SEQ ID NO:41, or SEQ ID NO:41 with a conservative amino acid substitution. In a particular embodiment the nucleic acid comprises a nucleotide sequence encoding a dimerization domain that has the amino acid sequence of SEQ ID NO:41. In a preferred embodiment of this type the isolated nucleic acid comprises the coding sequence of SEQ ID NO:40.
The present invention includes an isolated nucleic acid comprising a nucleotide sequence encoding a truncated vertebrate TRF that has the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:31 with a conservative amino acid substitution. In a particular embodiment the isolated nucleic acid comprises a nucleotide sequence encoding a truncated vertebrate TRF that has the amino acid sequence of SEQ ID NO:31. In a preferred embodiment of this type the isolated nucleic acid has the nucleotide sequence of SEQ ID NO:30.
The present invention also provides all of the peptides or proteins that are encoded by all of the nucleic acids of the present invention including isolated TRFs, proteolytic fragments of TRFs, truncated proteins, and peptides or proteins comprising a particular domain of a TRF such as the dimerization domain, the Myb domain or the basic or acidic N-terminal domain of the TRF. In addition, all of these proteins and peptides can be combined into corresponding chimeric proteins or peptides such as fusion proteins or fusion peptides. Such chimeric proteins and peptides are also part of the present invention including for example, a chimeric protein having an N-terminal domain and a dimerization domain of a TRF2 and a Myb domain of TRF1.
In one such embodiment the isolated vertebrate telomere repeat binding factor (TRF) has an amino acid sequence substantially homologous to that of SEQ ID NO:27, and comprises the following characteristics: a basic N-terminal domain, a dimerization domain, and a Myb domain. In a preferred embodiment when the basic N-terminal domain is removed the TRF detectably binds to the telomere repeat sequence (TTAGGG)12. Such binding is preferably detected in an in vitro assay. Preferably the isolated TRF is a is a mammalian protein.
In one embodiment the isolated TRF is a human protein having the amino acid sequence of SEQ ID NO:27, or SEQ ID NO:27 with a conservative amino acid substitution. In a particular embodiment the isolated TRF has the amino acid sequence of SEQ ID NO:27. In another embodiment the isolated TRF is a murine protein having the amino acid sequence of SEQ ID NO:29, or SEQ ID NO:29 with a conservative amino acid substitution. In a particular embodiment the isolated TRF has the amino acid sequence of SEQ ID NO:29.
The present invention further provides an isolated protein comprising the basic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:37, or SEQ ID NO:37 with a conservative amino acid substitution. In a particular embodiment the isolated the basic N-terminal domain has the amino acid sequence of SEQ ID NO:37. Another embodiment comprises the basic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:39, or SEQ ID NO:39 with a conservative amino acid substitution. In a particular embodiment the isolated the basic N-terminal domain has the amino acid sequence of SEQ ID NO:39.
An isolated protein comprising a dimerization domain of a TRF having the amino acid sequence of SEQ ID NO:45, or SEQ ID NO:45 with a conservative amino acid substitution is also included in the present invention. In a particular embodiment the isolated the dimerization domain has the amino acid sequence of SEQ ID NO:45. A related embodiment contains an isolated protein comprising a dimerization domain of a TRF having the amino acid sequence of SEQ ID NO:47, or SEQ ID NO:47 with a conservative amino acid substitution. In a particular embodiment the isolated the dimerization domain has the amino acid sequence of SEQ ID NO:47.
The present invention also includes an isolated protein comprising a dimerization domain of a TRF having the amino acid sequence of SEQ ID NO:41, or SEQ ID NO:41 with a conservative amino acid substitution. In a particular embodiment the isolated the dimerization domain has the amino acid sequence of SEQ ID NO:41. A related embodiment contains an isolated protein comprising a dimerization domain of a TRF having the amino acid sequence of SEQ ID NO:43, or SEQ ID NO:43 with a conservative amino acid substitution. In a particular embodiment the isolated the dimerization domain has the amino acid sequence of SEQ ID NO:43. In yet another embodiment an isolated protein comprises a dimerization domain of a TRF that has the amino acid sequence of SEQ ID NO:49, or SEQ ID NO:49 with a conservative amino acid substitution. In a particular embodiment of this type, the dimerization domain has the amino acid sequence of SEQ ID NO:49.
The present invention further provides an isolated avian TRF encoded by SEQ ID NO:24.
The present invention also provides an isolated protein that is a truncated TRF having the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:31 with a conservative amino acid substitution. In a particular embodiment the isolated the truncated TRF has the amino acid sequence of SEQ ID NO:31.
The present invention also includes antibodies to all of the TRFs and TRF domains of the present invention. One such embodiment is an antibody that recognizes a basic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:37. In another such embodiment the antibody recognizes a basic N-terminal domain of a TRF that has the amino acid sequence of SEQ ID NO:39.
Such antibodies can be polyclonal, monoclonal, and/or chimeric antibodies. The present invention also includes immortal cell lines that produce the monoclonal antibodies of the present invention.
In a related aspect of the present invention, a novel method for purifying telomeric binding proteins is provided, which comprises the steps of:
a) isolating nuclei from tissue culture cells;
b) preparing nuclear extracts of the nuclei;
c) contacting the nuclear extracts with an affinity chromatography column comprising a bound DNA fragment, wherein the DNA fragment comprises TTAGGG repeat sequences; and
d) eluting telomeric binding proteins from the column.
In a particular embodiment, casein is added to the eluted telomeric binding proteins to obtain active DNA-binding proteins.
In another aspect of the present invention, the TRFs of the present invention or antagonists or agonists thereof may be used to counteract the shortening of telomere length which occurs during aging, and to counteract the abnormal telomere physiology present in cancerous cells. Accordingly, methods of providing a TRF and/or its agonists or antagonists are contemplated.
Still a further aspect of the present invention extends to antibodies and oligonucleotide probes to the TRFs of the present invention, which may be used for both diagnostic and therapeutic approaches.
The DNA sequences of the TRFs of the present invention or portions thereof, may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species. The present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the TRF. For example, the probes may be prepared with a variety of known vectors, such as the phage xcex vector. The present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences in the present invention. Correspondingly, the preparation of antisense RNA and ribozymes are included herein.
The present invention also includes TRF proteins having the activities noted herein, and that have the amino acid sequences included in the present invention.
The present invention provides specific factors i.e., TRFs, which bind to TTAGGG repeat sequences as described earlier. Accordingly, the exact structure of each TRF will understandably vary so as to achieve this DNA binding and activity specificity. It is this specificity and the direct involvement of the TRF in the chain of events leading to telomere length regulation, that offers the promise of a broad spectrum of diagnostic and therapeutic utilities.
The present invention naturally contemplates several means for preparation of a TRF, including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope. The isolation of the cDNAs that encode a TRF amino acid sequence disclosed herein (e.g., using a nucleic acid that hybridizes with the cDNA encoding a TRF to act as a probe/binding partner to detect and/or isolate a nucleic acid encoding a related TRF), facilitates the reproduction of the TRF by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
The invention includes an assay system for screening of potential drugs effective to modulate TRF activity of target mammalian cells by interrupting or potentiating the activity of the TRF. In one instance, the test drug could be administered to a cellular sample containing the TRF along with telomeric sequences, to determine its effect upon the binding activity of the TRF to the DNA, or to the test drug, by comparison with a control. In still another assay, the purified TRFs or a particular structural/functional domain are used as targets for testing the binding characteristics of potential drugs. For example, the basic N-terminal domain of TRF2 and acidic N-terminal domain of TRF1 can be employed to screen potential drugs for binding specificity for the two corresponding TRFs. In this way a drug can be readily identified which is likely to interfere with TRF1 without interfering with TRF2.
The assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to a TRF and/or other telomeric binding factors or proteins in the nucleus, thereby inhibiting or potentiating the activity of the TRF. Such assays would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity. For example, such drugs might be used to inhibit the proliferation of cells in cancerous states, or to treat cells which are aging, or to treat other pathologies associated with variations in telomere length.
In yet a further embodiment, the invention contemplates antagonists of the activity of a TRF, in particular, an agent or molecule that inhibits the role of TRFs in telomere function. In a specific embodiment, the antagonist can be a peptide having the sequence of a portion of a DNA binding domain of a TRF, such as that illustrated by SEQ ID NO:13.
The diagnostic utility of the present invention extends to the use of the present TRF in assays to screen for cancer and other inherited diseases associated with telomere length.
The present invention likewise extends to the development of antibodies against the TRFs or to the specific structural/functional domains of the TRFs of the present invention, including naturally raised and recombinantly prepared antibodies. For example an antibody raised against the basic N-terminal domain of a TRF can be used to distinguish a TRF2 from a TRF1. In addition, the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the TRFs. Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating telomere length.
Thus, the TRFs, their analogs and/or agonists, and any antagonists or antibodies that may be raised thereto, are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the TRF that has been labelled by either radioactive addition, reduction with sodium borohydride, or radio iodination.
In an immunoassay, a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labelled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labelled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached.
In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re are used, known currently available counting procedures may be utilized. In the instance where the label is an enzyme, detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
The present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the TRF, or to identify drugs or other agents that may mimic or block their activity. The system or test kit may comprise a labelled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the TRFs, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labelled component, its binding partner, one of the components to be determined or their binding partner(s).
In a further embodiment, the present invention relates to certain therapeutic methods which would be based upon the activity of the TRF(s), its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity. A first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the binding activity of the TRF or its subunits, and comprises administering an agent capable of modulating the production and/or activity of the TRF or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host. For example, drugs or other binding partners to the TRF or proteins may be administered to inhibit or potentiate TRF activity, as in the potentiation of TRF activity in aging, or the inhibition or modulation of TRF activity in cancer therapy.
More specifically, the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of activation of the TRF or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention. For example, drugs or other binding partners to the TRF or proteins, as represented by SEQ ID NO:12, may be administered to inhibit or potentiate telomere lengthening activity, as in the potentiation of TRF in cancer therapy.
In particular, the isolated TRFs, proteolytic fragments of TRFs, truncated proteins, and peptides or proteins which comprise a particular structural/functional domain of a TRF, their antibodies, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances where appropriate, such as to treat cancer or counteract the aging process.
Accordingly, it is a principal object of the present invention to provide TRFs in purified form that exhibits certain characteristics and activities associated with telomere lengthening activity.
It is a further object of the present invention to provide antibodies to the TRFs, and methods for their preparation, including by recombinant means.
It is a further object of the present invention to provide a method for detecting the presence of the TRF and its subunits in mammals in which invasive, spontaneous, or idiopathic pathological states are suspected to be present.
It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, potentially effective in either mimicking the activity or combating the adverse effects of the TRFs and/or its subunits in mammals.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the TRF or subunits thereof, so as to alter the adverse consequences of such presence or activity, or where beneficial, to enhance such activity.
It is a still further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the TRF or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous or idiopathic pathological states.
It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the TRF, its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of the TRF.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with reference to the following illustrative drawings.